This is the competitive renewal of the Drosophila Genome Center. The Center proposes to move forward in its characterization of the Drosophila genome in three major areas: (1) physical mapping; (2) gene discovery, gene disruption, and other biological annotation of the genome map; an (3) large-scale genomic sequencing. By the end of the first funding period, the physical map will provide nearly complete coverage of the 120 megabase euchromatic portion of the Drosophila melanogaster genome. The focus of the proposed physical mapping work is on gap-filling and contig closure. The impetus behind the biological annotation experiments is our conviction that the complete Drosophila sequence will be a fundamental tool in the next century. Towards this end one of the goals of this grant is to develop set of biological and informatics tools, reagents, and an informational database to facilitate the biologically meaningful interpretation of the Drosophilia genomic sequence. For example, we are carrying out gene disruption experiments on a scale - inactivating more than 25% of all vital genes - that is unprecedented in multicellular organisms. This work will guarantee that as large scale sequencing of Drosophila and human genomes progresses, a significant fraction of human genes with Drosophila homologues will be associated with transposable element induced mutations that can provide insight into their function during Drosophila and human development. The cornerstone of this proposal is the determination of the DNA sequence of the euchromatic portion of the Drosophila genome. We present the next five years of a plan that should, shortly after the turn of the century, lead to the complete elucidation of the genomic sequence for this central model organism. The degree of evolutionary conservation - not just of gene sequence, but also of gene function - that has emerged from the study of genes and pathways in different organisms over the last few years has surprised even the most avid proponents of model organism research. Just as the Rosetta stone gave the world the key to the long-forgotten language of ancient Egypt, so the biologically-annotated sequences of the genomes of the model organisms, and in particular Drosophila, will provide an invaluable key to understanding the sequence of the human genome. A major goal of the Drosophila Genome Center is to facilitate the endeavor.